Ikei Kobayashi

Increased Frequency of Surface IgA-Positive Plasma Cells in the Intestinal Lamina Propria and Decreased IgA Excretion in Hyper IgA (HIGA) Mice, a Murine Model of IgA Nephropathy with Hyperserum IgA

Because abnormalities of mucosal immunity have been suggested in human IgA nephropathy, we examined the involvement of mucosal immunity in IgA deposition to the kidney in hyper IgA (HIGA) mice, which was established as a mouse model for human IgA nephropathy with hyperserum IgA. The number of surface IgA+B220− lymphocytes in the intestinal lamina propria (LP) of HIGA mice increased 2.7-fold at 30 wk of age as compared with those at 10 wk of age, whereas normal mice did not show such increase. The surface IgA+B220− LP lymphocytes spontaneously secreted IgA in culture. Morphological studies showed that the surface IgA+B220− lymphocytes of murine intestinal LP are identical with plasma cells (PCs). About 20% of IgA+B220− PC in LP expressed both Mac-1 and CD19, suggesting that they may derive from peritoneal B-1 cells. Cell cycle study on intestinal IgA-PCs using bromodeoxyuridine revealed no difference between HIGA mice and normal mice, suggesting that the high frequency of IgA-producing PCs in HIGA mice is not due to enhanced proliferation or prolonged survival of IgA-producing PCs in LP. In addition, IgA secretion into the gut lumen of HIGA mice decreased drastically (to one forth) with aging. These data suggest that the increased number of intestinal IgA-producing PCs and the down-regulation of IgA excretion into the intestinal lumen might synergistically contribute to the hyperserum IgA in HIGA mice and resultant IgA deposition to the kidney.

Roles of coagulation pathway and factor Xa in rat mesangioproliferative glomerulonephritis.

Tissue factor initiates the extrinsic coagulation pathway by activating coagulation factor X to factor Xa, and factor V is a cofactor for the prothrombin activation by factor Xa. As factor Xa is known to promote the proliferation of mesangial cells in culture, the roles of the coagulation pathway and factor Xa were studied in an animal model of mesangioproliferative glomerulonephritis (MsPGN). MsPGN was induced in Wistar rats by an intravenous injection of anti-Thy 1.1 monoclonal antibody, OX-7. To clarify the role of factor Xa in MsPGN, a specific factor Xa inhibitor, DX-9065a, was injected intravenously at 2.5 or 10 mg/kg at the same time as OX-7, and kidney involvement was assessed by immunohistological analyses. We also examined p44/42 mitogen-activated protein (MAP) kinase activation. Time-course study revealed that expressions of tissue factor, factor V, and protease-activated receptor 2 (PAR2) were peaked on day 3, followed by factor X accumulation and mesangial proliferation. DX-9065a treatment significantly ameliorated proteinuria in a dose-dependent manner on day 8. Histological analyses showed a significant reduction in the size of glomeruli, the total number of glomerular cells, and crescent formation by DX-9065a treatment. Macrophage infiltration, which was rapidly observed on day 1 in disease control rats was not inhibited on days 1–3 by DX-9065a treatment, however it was suppressed on days 5–8. The deposition of fibrin, the number of PCNA-positive cells, and phosphorylation of p44/42 MAP kinase were markedly increased in the disease control group, whereas they were significantly reduced in the treatment group. Tissue factor and factor V induction may accelerate MsPGN through the activation and accumulation of factor X via proinflammatory and procoagulant mechanisms, and the inhibition of factor Xa would be a promising method to regulate the disease process.

Quantitative trait loci (QTL) analysis reveals a close linkage between the hinge region and trimeric IgA dominancy in a high IgA strain (HIGA) of ddY mice

Polymerization of IgA has been suggested as one of the causes of mesangial deposition in IgA nephropathy. HIGA mice are an inbred model of IgA nephropathy, established by selective mating of ddY mice. This strain is characterized by a unique profile of the IgA molecule that is dominantly polymeric and has high serum levels with intense IgA deposition on the mesangium. We carried out quantitative trait loci (QTL) analysis, using F2 generations by crossing HIGA with BALB/c mice. Significant linkage of polymeric IgA in serum samples was identified around D12Mit263, which is close to the gene of the immunoglobulin heavy chain on chromosome 12. The amino acid sequence of the α heavy chain revealed marked differences between BALB/c and HIGA mice. Furthermore, most differences were focussed on the hinge region. The DBA/2J strain, which has the same amino acid sequence in the hinge region as the HIGA strain, also showed polymeric IgA dominance but low IgA levels in sera. Size fraction analysis revealed that these polymeric IgA showed trimer dominance in both DBA/2J and HIGA mice. In conclusion, the hinge region plays a key role in trimeric IgA formation in HIGA mice.

Up-Regulated TGF-β mRNA Expression in Splenic T Cells of High IgA-Prone Mice: A Murine Model of IgA Nephropathy with Glomerulosclerosis

Background/Aims: Recently, we established a high serum IgA-prone inbred (HIGA) mouse strain as a murine model of spontaneous IgA nephropathy by selective mating of high serum IgA ddY mice, and found that they showed enhanced production of glomerular extracellular matrix components with increased expression of TGF-β mRNA and protein in the kidneys. In this study, we examined the roles of lymphocytes in the development of high serum IgA in this strain. Methods: We performed flow cytometric analyses of T and B cells in splenic mononuclear cells (SMNCs) from these mice using BALB/c mice as normal controls. We also compared serum TGF-β1 concentrations and TGF-β mRNA expression levels in the B-cell-depleted (T-cell-rich) fraction of SMNCs in these mice. Results: HIGA mice showed significantly fewer CD3-positive cells compared with BALB/c mice when young, but not when aged. The CD4/CD8 ratio of HIGA mice was lower than that of BALB/c mice, but this difference was not significant. Although the number of B220-positive cells did not vary significantly, the ratio of surface IgA-positive B cells was significantly increased in both young and adult HIGA mice. The B-cell-depleted SMNCs from HIGA mice exhibited higher levels of expression of TGF-β and TGF-β1 mRNA than controls from a young age, which were maintained throughout life, but there were no differences in PDGF, MCP-1 or bFGF expression between these two strains. In contrast to local mRNA expression, serum TGF-β1 concentration was decreased in HIGA mice compared with BALB/c controls. Conclusion: These findings suggest that the mating procedure performed to establish HIGA mice selected for a unique phenotype of local up-regulation of TGF-β production in the kidneys, as well as T cells that may contribute to both the early and consistently high serum IgA expression and enhanced production of renal extracellular matrix components in HIGA mice. Additionally, TGF-β1 may act locally, not systemically, in a paracrine or autocrine manner.

Role of transforming growth factor-β/Smad signalling pathway in enhanced production of glomerular extracellular matrix in IgA nephropathy of high-serum-IgA ddY mice

SUMMARY: The high‐IgA inbred strain (HIGA) of ddY mice, an animal model of human IgA nephropathy, shows consistently high serum IgA levels, progressive mesangial sclerosis and IgA deposition, and elevated renal expression of transforming growth factor (TGF)‐β. In the present study the role of the TGF‐β/Smad signalling pathway in extracellular matrix (ECM) production was assessed in cultured mesangial cells derived from HIGA mice. The production of type I and type IV collagens in response to TGF‐β1, expression of Smad2, Smad4, Smad7, and Sp1 and p300, and phosphorylation of Smad2 by TGF‐β1 were assessed in cultured mesangial cells derived from HIGA mice at the age of 10 weeks by comparison with age‐matched C57BL/6 mice as controls. In addtion, the expression of p300 and type I and type IV collagens in renal tissues of HIGA mice at the age of 60 weeks was determined. The production of type I and type IV collagens by cultured mesangial cells in HIGA mice was markedly upregulated compared with that in C57BL/6 mice. Although protein expression levels of Smad2, Smad4, Smad7, and Sp1 in the mesangial cells were similar in the two mouse strains, upregulation of p300 was marked in HIGA mice. Expression of p300 in renal tissues of HIGA mice was also enhanced in HIGA mice when compared with C57BL/6 mice. In HIGA mice, p300 expression was upregulated in the mesangial cells both in vitro and in vivo. It appears that the upregulation of p300 may be related to glomerular sclerosis associated with IgA nephropathy in HIGA mice.

Preeclamptic nephropathy associated with membranous nephropathy

We report a patient who had nephropathy of toxemia of pregnancy associated with membranous glomerulonephritis, which deteriorated 3 months after delivery. Electron microscopy revealed electron-dense deposits on the subepithelial surface of the glomerular basement membrane and finely granular/fibrillar materials in the subendothelial space. As a result of treatment with prednisolone and anticoagulants, marked alleviation of both proteinuria and edema was achieved. We therefore consider that the change from subclinical to clinical membranous nephropathy could be attributed to pregnancy.

Genetic analysis in a high IgA strain of ddY (HIGA) mice

Introduction: HIGA mouse is an inbred model of IgA nephropathy (IgAN), established by selective mating of ddY mice bearing high serum IgA. HIGA mouse is characterized with peculiar IgA characters similar to human IgAN, markedly high serum levels, dominantly polymeric sizes and low ratios of sialylation and galactosylation. Mesangiosclerotic lesions with IgA deposition are advanced with aging. In the present study, to investigate the genetic background of IgA abnormality in this mouse, we disclosed the form of inheritance of these abnormal IgA characters and investigated the contribution of these characters to the renal lesion especially to the mesangial IgA deposition. Methods: For the genetic analysis of inheritance, F1 mice mated between HIGA and BALB/c mice (chosen as controls) were produced. Serum IgA levels, molecular size and glycosylation were determined in 30-week-old BALB/c, HIGA and F1 mice by ELISA, Western blot and lectin binding assays, respectively. In Western blot, the samples were diluted to 50–500 times and after 6% SDSPAGE in the non-reducing condition, IgA staining was visualized by ECL systems. Signal intensity was measured by NIH image and molecular size was expressed as polymer/dimer ratios (P/D). Lectin binding assays were designed using biotinylated sambucus nigra bark lectin (SNA) which recognizes sialic acid. Plates coated with goat anti-mouse IgA were incubated with the samples, and then biotinylated SNA was applied. After alkalinephosphatase-conjugated streptavidin was applied, the binding lectin was measured as well as IgA. Each obtained value was divided by each IgA concentration. Furthermore, to analyze the relationship between IgA characters and the renal lesions, 34 F2 (F1 ¥ F1) mice were produced. Serum levels, molecular size and sialylation of IgA were examined in 40-week-old F2 mice. IgA staining in frozen renal sections (2 mm) was performed using FITC-conjugated anti-IgA antibody. Intensity of glomerular IgA was expressed as mean brightness calculated from 0–255 by Photoshop in five glomeruli of each mouse. Multiregression analyses were applied for the examination of correlation among these factors. Results: In 30-week-old mice, compared with original HIGA mice, serum IgA levels of F1 mice were significantly suppressed (14.8 ± 18.54 in BALB/c mice, 182 ± 87.57 in HIGA mice and 54.2 ± 21.22 in F1 mice). Western blot analysis showed monomeric and dimeric IgA dominance in BALB/c mice while it showed polymer IgA dominance in HIGA mice. F1 mice showed dimeric IgA dominance with a little monomeric IgA, which resembled the pattern of BALB/c mice. Ratio of SNA-bound IgA was significantly lower in HIGA mice than in BALB/c mice (0.19 ± 0.003 in BALB/c vs 0.16 ± 0.002 in HIGA), while the analysis in F1 mice resembled the pattern of HIGA mice (0.15 ± 0.002). These results indicate that in HIGA mice the traits of high IgA, polymeric IgA dominance might be regulated by recessive genes, on the other hand, that of low ratio sialylated IgA might be regulated by dominant genes. In 40-week-old F2 mice, the analysis of the relationship between serum IgA levels and P/D ratio revealed no apparent correlation, indicating that these characters were independently regulated by different gene clusters. The multiregression analysis in F2 mice revealed that degree of mesangial IgA deposition was most tightly related with serum IgA levels (P = 0.0014), then with P/D ratio (P = 0.0620) and faintly with ratio of SNAbound IgA. Conclusion: Representative, physicochemical characters of IgA in HIGA mice, high serum levels, polymer dominancy, and underglycosilations, are independently regulated by recessive and dominant genes and contribute to mesangial IgA deposition with different strength.